Process for the preparation of the 14β-hydroxy-baccatin III-1,14-carbonate

ABSTRACT

A process for the preparation of 14β-hydroxy-baccatin III-1,14-carbonate useful for the preparation of novel taxane derivatives with antitumor activity.

The present invention relates to a process for the preparation of14β-hydroxy-1,14-carbonate-baccatin III. The product obtained with theprocess of the invention can be used in the preparation of novel taxanederivatives with antitumor activity.

Taxanes are one of the most important classes of antitumor agentsdeveloped in recent years. Paclitaxel is a diterpene complex obtainedfrom the bark of Taxus brevifolia and is considered one of the majormedicaments for the therapy of cancer. At present, an extensive searchis being carried out for novel taxane derivatives having superiorpharmacological activity and improved pharmacokinetic profile. Aspecific approach relates to baccatin III derivatives variously modifiedwith respect to the parent structure. Examples of said compounds arerepresented by the 14β-hydroxy baccatin III derivatives disclosed inU.S. Pat. No. 5,705,508, WO 97/43291, WO 96/36622. At present,14β-hydroxy-deacetylbaccatin III 1,14-carbonate derivatives are preparedstarting from the precursor 14β-hydroxy-deacetylbaccatin III, which is anatural compound obtainable in small amounts by extraction of the leavesof Taxus wallichiana, as disclosed in EP 559019. There is strong needfor novel processes for the easy, effective preparation of large amountsof 14β-hydroxy-1,14-carbonate-baccatin III, and hence the derivativesthereof.

It has now been found that 14β-hydroxy-baccatin III-1,14-carbonate canbe prepared with a process starting from 13-ketobaccatin III, whichcompound can be easily obtained from 10-deacetylbaccatin III, which canin turn be easily isolated in large amounts from the leaves of Taxusbaccata, contrary to 14β-hydroxy-baccatin III.

Therefore, the invention relates to a process for the preparation of14β-hydroxy-baccatin III-1,14-carbonate which comprises the followingsteps:

a. treatment of 7-Boc-13-ketobaccatin III of formula

with suitable bases and oxidizing agents, to give7-Boc-13-keto-14-hydroxy-baccatin III:

b. carbonation of the 1 and 14 hydroxy groups to give14β-Hydroxy-7-Boc-13-keto-baccatin III-1,14-carbonate:

c. reduction of the ketone at the 13 position and cleavage of theprotective group in 7 or vice versa.

Starting 13-ketobaccatin III is conveniently protected at the 7 positionwith a suitable protective group, preferably tert-butoxycarbonyl (Boc).Step a) is carried out by treatment with a suitable base, in particularpotassium t-butoxide (t-BuOK) or potassium bis(trimethylsilyl)amide(KHMDS). The reaction can be carried out at −40 to −78° C. Suitablesolvents for this reaction are ethers, such as tetrahydrofuran ordiethyl ether, in particular in mixture with hexamethylphosphoramide(HMPA) or 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)pyrimidinone (DMPU). Theenolate is then treated with an oxidizing agent, such as oxaziridinederivatives (in particular N-benzenesulfonyl phenyl oxaziridine,N-benzenesulfonyl m-nitrophenyl oxaziridine andcamphorsulfonyloxaziridine) to provide the 7-protected13-keto-14-hydroxy-baccatin III derivative.

Step b) is then carried out by treatment with a carbonylating agent (forexample carbonyldiimidazole or phosgene) under the conditions usuallydescribed in literature, to provide the 1,14-carbonate derivative. Thereaction can be conveniently carried out in inert solvents, preferablyethers or chlorinated solvents, in the presence of a base (preferablypyridine or triethylamine), at a temperature ranging from −40° C. toroom temperature. The reaction can be carried out both on the purestarting material and on the crude from the previous step.

The reduction of the carbonyl at the 13 position of step c) is easilycarried out with tetrabutylammonium borohydride in ethanol at atemperature usually ranging from −20 to −50° C., and is completed within2-6 hours. The reaction can also be carried out in methanol,isopropanol, or in a methanol and tetrahydrofuran mixture. The reducingagent can be used in stoichiometric amount, although a hydride excess ispreferably used. The reduction can also be effected with other hydrides,preferably lithium borohydride, sodium borohydride, sodium triacetoxyborohydride, in the conditions known in the technique.

Protection at the 7 position is removed under conditions depending onthe protective group used. For example, if the protective group at the 7position is tert-butoxycarbonyl, hydrolysis with formic acid cansuccessfully be used.

The starting 13-ketobaccatin III can be readily prepared according toone of the two following procedures.

10-Deacetyl-baccatin III is selectively oxidized at the 13-position withozone to give 13-keto-10-deacetyl baccatin III. Oxidation can be carriedout in alcoholic or chlorinated solvents, in particular methanol ormethylene chloride, at a temperature ranging from −78 to roomtemperature. 13-Keto-10-deacetyl-baccatin III is then regioselectivelyacetylated to give 13-keto-baccatin III.

Alternatively, 13-keto-baccatin III can be obtained by oxidation ofbaccatin III either natural or obtainable by regioselective acetylationof 10-deacetylbaccatin III. Oxidation can be carried out with ozone, oralso with manganese dioxide in aprotic solvents such as methylenechloride, at temperatures ranging from 0° C. to 60° C., more preferablyat room temperature.

The processes of the invention are summarized in the following scheme:

The following examples further illustrate the invention.

The abbreviations used are as follows:

AcOEt=ethyl acetate; TES=triethylsilyl; TESCl=triethylsilyl chloride;DCM=dichloromethane, THF=tetrahydrofuran, HMPA=hexamethylphosphoramide,DMPU=1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)pyrimidinone.

EXAMPLE 1 10-Deacetyl-13-keto-baccatin III

10-Deacetyl-baccatin III (3 g, 5 mmol) was dissolved in DCM-MeOH 1:1(250 ml) and cooled to −78° C. An ozone stream (1.4 g/ml) was bubbledthrough the solution until disappearance of the starting material (2 h).The ozone stream was replaced with nitrogen. The solution was thentreated with dimethyl sulfide (1 ml) and pyridine (1 ml), the solventwas evaporated off and the crude was dissolved in EtOAc (100 ml) andwashed with 0.1 N HCl and ice. After evaporating off the solvent, thetitle product was obtained in a 90% yield.

EXAMPLE 2 13-Keto-baccatin III

Baccatin III (150 g, 0.25 mol) was dissolved in acetone (1.43 I).Commercially available manganese dioxide (450 g) was added in threeportions under strong stirring. After the starting product disappeared(4 h) the suspension was filtered and the solvent evaporated off. Thecrude was suspended in EtOAc (100 ml) and refluxed for 1 h, then c-Hex(100 ml) was added. The title compound was obtained from mother liquors,after evaporation of the solvent, as white solid (140 g, 95%).

EXAMPLE 3 7-Boc-13-keto-baccatin III

A solution of 13-keto-baccatin III (1.1 g, 1.9 mmol) in methylenechloride (0.5 ml) was dissolved in carbon tetrachloride (14 ml) at roomtemperature. 1-methylimidazole (35 ml, 0.282 mmol) and Boc₂O (1.026 g,4.7 mmol) were then added under stirring strong. The solution was leftunder stirring at 20° C. for 18 hours. After that, the solvent wasreplaced with acetone (5 ml), the solution was poured into water (5 ml)and left under stirring overnight. The precipitate was collected onbuchner funnel, washed with n-pentane and dried to give 1.1 g of thetitle product (1.78 mmol, 94%).

EXAMPLE 4 14-Hydroxy-7-Boc-13-ketobaccatin III

A solution of 7-Boc-13-keto-baccatin III (0.65 g, 0.95 mmol) in THF-DMPU8:2 (10 ml) was added to a solution of t-BuOK (0.425 g, 3.79 mmol) inanhydrous THF (10 ml) under stirring at −60° C. After 15 minutes, asolution of camphorsulfonyloxaziridine (2.63 mmol) in THF-DMPU 8:2 (10ml) was added. After the starting material disappeared (45 min), thereaction was quenched with glacial acetic acid (0.4 ml) and the mixturewas diluted with 10% aqueous NH₄Cl al (25 ml). The organic layer waswashed with water, dried over sodium sulfate and evaporated underreduced pressure. The product was used for the subsequent step withoutpurification.

EXAMPLE 5 14β-Hydroxy-7-Boc-13-ketobaccatin III 1,14-carbonate

14-Hydroxy-7-Boc-13-ketobaccatin III (2.0 g) and carbonyldiimidazole(0.65 g, 4.0 mmol) were dissolved in toluene (11 ml) and heated at 75°C. under stirring for 90 min. The solution was cooled to roomtemperature and treated with 0.2 N HCl (5 ml). The organic layer wasdiluted with EtOAc (15 ml), washed with water, dried, and the solventwas evaporated off. The title compound was obtained byflash-chromatography (silica gel, cHex/DCM/Et₂O, 14:3.5:2.5) as a whitesolid (0.87 g, 1.20 mmol, 82% on two steps).

EXAMPLE 6 14β-Hydroxy-7-Boc-baccatin III 1,14-carbonate

A solution of 14β-Hydroxy -7-Boc-13-ketobaccatin III 1,14-carbonate inTHF (3 ml) was added to a solution of tetrabutylammonium borohydride(1.29 g) in dry methanol (11 ml) at −50° C. under inert atmosphere.After 4 hours the reaction was quenched with a solution of citric acid(1.5 g) in water (5 ml). The mixture was extracted with ethyl acetate.The organic phase was dried over magnesium sulfate and evaporated underreduced pressure. The crude was purified by column chromatography togive 14β-Hydroxy-7-Boc-baccatin III 1,14-carbonate (68%) and13-epi-14β-Hydroxy -7-Boc-baccatin III 1,14-carbonate (28%) in a 70%conversion yield.

EXAMPLE 7 14β-Hydroxy-baccatin III 1,14-carbonate

A 97% formic acid solution (5 ml) was added to a solution14β-Hydroxy-7-Boc-baccatin III 1,14-carbonate (0.50 g, 0.68 mol) indichloromethane (3 ml) at −8° C. The reaction was kept under stirringfor 5 days, then neutralized with 2N ammonia. The organic phase wasextracted with ethyl acetate, dried and evaporated under reducedpressure. Silica gel chromatography (hexane-ethyl acetate=1.0:1.3)afforded the product as a white solid in a 65% yield.

1. A process for the preparation of 14β-hydroxy--l,14-carbonate-baccatinIII, which comprises: a) treatment of 7-Boc-13-ketobaccatin III offormula

with suitable bases and oxaziridine compounds as oxidizing agents, togive 7-Boc-13-keto-14-hydroxy-baccatin III:

b) carbonation of the 1 and 14 hydroxy groups to have14β-Hydroxy-7-Boc-13-keto-baccatin III-1,14-carbonate:

c) reduction of the ketone at the 13 position and cleavage of theprotective group in
 7. 2. The process as claimed in claim 1, whereinstep a) is carried out by treatment with potassium t-butoxide orpotassium bis(trimethylsilyl)amide at a temperature from -40 to -78° C.in ethers in mixture with hexamethylphosphoramide (HMPA) or 1,3-dimethyl-3,4,5, 6-tetrahydro-2(1H)pyrimidinone (DMPU), in the presenceof oxaziridine compounds.
 3. The process as claimed in claim 1, whereinstep b) is carried out by treatment with a carbonyldiimidazole orphosgene in chlorinated solvents in the presence of a base attemperatures ranging from -40° C. to room temperature.
 4. The process asclaimed in claim 1, wherein step c) is carried out by treatment with ahydride at a temperature from -20 to -50° C.
 5. The process as claimedin claim 4 wherein the hydride is selected from the group consisting oftetrabutylammonium borohydride, tetraethylammonium borohydride, sodiumborohydride, lithium borohydride, and sodium triacetoxy borohydride, andwherein the reaction is carried out in ethanol, methanol, isopropanol,or in a methanol and tetrahydrofuran mixture.
 6. The process as claimedin claim 1, wherein 13-keto-baccatin III protected at the hydroxyl in 7is prepared by selective acetylation of the hydroxyl 10 followed byoxidation of the hydroxyl 13 and protection of the hydroxyl
 7. 7. Theprocess as claimed in claim 6, wherein 13-keto-baccatin III is obtainedby selective acetylation of deacetylbaccatin III in 10 with aceticanhydride followed, by oxidation with manganese dioxide in aproticsolvents at 0° C.-60° C. or by oxidation of baccatin III with ozone. 8.An intermediate compound 7-Boc-13-keto-14-hydroxy--baccatin III, offormula


9. An intermediate compound 14β-Hydroxy-7-Boc-13-keto-baccatinIII-1,14-carbonate, of formula:


10. An intermediate compound 14β-Hydroxy-7-Boc-13-hydroxy-baccatinIII-1,14-carbonate, of formula: